Plants have some specific metabolic pathways to synthesize a large number of valuable proteins [1]. Some toxic proteins produced by plants play an important role in defense responses against pathogens attack. For example, ribosome inactivating proteins (RIPs) are important anti-pathogenic proteins. Furthermore, RIPs are widely distributed throughout the kingdom of plants [2,3]. RIPs could function as N-glycosidases to remove a specific adenine residue from the highly conserved α-sarcin loop of large eukaryotic and prokaryotic rRNAs, thus arresting protein synthesis [4]. Various RIPs were reported successively to have broad spectrum antiviral activity against plant and animal viruses, anti-tumor activity, ribonuclease and deoxyribonuclease [5,6]. As for plants, genetic, physiological, and molecular analyses have revealed that RIPs have antiviral, antifungal, and insecticidal properties using biotechnology methods [7-9]. Pokeweed antiviral protein (PAP) is members of the type I RIPs that are isolated from the extracts of pokeweed plant [10]. Previous studies suggest that PAP causes a concentration-dependent depurination of HIV-1 [11], Tobacco mosaic virus (TMV) [12], tobacco etch virus (TEV) RNAs [13].

Plants are constantly attacked by pathogens, including bacteria, fungi, and viruses. Virus disease and pathogenic fungi have been always a major problem in the cultivation of many vegetable and ornamental crops all over the world. However, plants have evolved an array of natural defense strategies to defend these pathogens attack [14]. Systemic acquired resistance is characterized by micro-oxidative bursts, accumulation of salicylic acid (SA), and expression of pathogenesis-related (PR) genes in systemic tissues. Several studies suggest that RIPs may participate in the process of plant defense response against various pathogens attack. For example, our studies reveal that alpha-momorcharin, a RIP produced by bitter melon enhances plant systemic resistance against diverse plant viruses in tobacco plants [15]. Previous studies suggest that high levels of reactive oxygen species (ROS) cause cell death inside the cell. Our studies also suggest that exogenous application of PAP enhances plant systemic resistance to TMV infection by regulating ROS levels in Nicotiana benthamiana [16]. Three type of PAPs have been purified from the different seasons leaves of pokeweed plant (Phytolacca americana), and they all exhibit antiviral activity against plant and animal viruses [17-23]. Such as, the named PAP was purified from the spring leaves of pokeweed plant. The protein purified from the summer leaves of pokeweed plant was named PAP-II. And the called PAP-Ⅲ was purified from the early autumn leaves of pokeweed plant. In order to further investigate the role of PAP in plant systemic resistance response against TMV, transient over-expression biotechnology was used to express PAP-II in N. benthamianaplants in this report.

Materials and Methods

Plant growth conditions and pathogen inoculation

Wild-type Nicotiana benthamiana plants were grown in a greenhouse at 25°C with a 16-h-light/8-h-dark cycle (100 μmol m-2s-1). Six- to seven-week-old seedlings were used in the experiments. For Tobacco mosaic virus (TMV) inoculation,Agrobacterium strain GV3101 cultures at OD600 = 0.5 containing the TMV-GFP construct were infiltrated into the secondary leaves [24].

Cloning of PAP-II gene from Phytolaccaamericana by RT-PCR

Previous studies suggest that the PAP-II gene has been successfully cloned from the summer leaves of pokeweed plant [22]. In this study, We also cloned the full-length PAP-II gene from the summer leaves of pokeweed plant by RT-PCR using the specific primers PAP-II-F (5’-ATGAAGATGAAGGTGTTA-3’) and PAP-II-R (5’-TCACTCGAATTCACCAA-3’) according to the reported sequence of PAP-II gene (GenBank accession X78628). And then the PCR product was sent to Invitrogen (Shanghai China) for sequencing analysis.

RNA extraction and quantitative real-time PCR

Total RNAs were isolated from N. benthamiana leaves as previously described using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. [24]. In order to detect the TMV accumulation levels, the upper leaves upon the secondary leaves (infiltration with Agrobacterium cultures containing the TMV-GFP construct) were collected. The first-strand cDNA was prepared using the ReverTra Ace kit (Toyobo Co., Ltd., Osaka, Japan). To further assay the expression levels of genes, quantitative real-time PCR analysis was performed on a Bio-Rad iCycler (Bio-Rad, Beijing, China). The qRT-PCR analysis was performed with the primers shown in Table 1. Relative quantitation of the target gene expressionlevel was performed using the comparative Ct (threshold cycle) method [15]. Three technical replicates were performed, each experiment included at least three independent plants. Amplification of ACTIN gene was used as an internal control.

The ORF of PAP-II was amplified by RT-PCR using the specific primers PAP-II-F’ (5’-GCTCTAGAATGAAGATGAAGGTGTTA-3’) and PAP-II-R’ (5’-CGCGGATCCTCACTCGAATTCACCAA-3’), which incorporate restriction sites for XbaI and BamHI at the product ends. Then the full-length sequence of PAP-II gene digested by XbaI and BamHI was cloned into the binary vector pBI121 digested by the same restriction enzymes under the control of the CaMV35S promoter (Figure 2A). Agrobacterium– mediated transient expression assay was performed as described previously [25]. The binary plasmids were transformed intoAgrobacterium tumefaciens strain GV3101 by electroporation. Transient expression assays were performed in at one site on the primary leaf by infiltration with Agrobacterium strain GV3101 containing the empty and the PAP-II-expressing vectors. Then the virus infection assays were performed in the 35S: PAP-II and control (35S: 00) plants 48 h after agro-infiltration.

Protein extraction and Western blot analysis

The 35S: 00 or 35S: PAP-II-inoculated leaves were used for western blotting. Total proteins were extracted with extraction buffer (50 mM Tris-Cl [pH 6.8], 5% mercaptoethanol, 10% glycerol, 4% sodium dodecyl sulfate, and 4 M urea) in an ice bath. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard [26]. The antibody used in the experiment was purchased from Hongze Biotechnology (Yangzhou China). Western blot analysis was performed according to Zhu et al. [15].

Statistical Analysis

At least three technical replicates were used in each determination. Values were expressed as mean ± SD. The results were statistically analyzed using one-way analysis of variance (ANOVA) and tested for significance (p<0.05) using Duncan’s test.

Results and Discussion

Cloning of PAP-II gene from Phytolacca americana

In order to clone the ORF of PAP-II gene, total RNAs were isolated from the summer leaves of pokeweed plant. As shown inFigure 1, we successfully obtained an approximately 930 bp fragment by RT-PCR using the specific primers PAP-II-F and PAP-II-R according to the reported sequence of PAP-II gene. Sequencing analysis demonstrated that the fragment, approx. 930 bp was the PAP-II gene. Therefore, we successfully cloned the ORF of PAP-II gene from the summer leaves of pokeweed plant.

Our previous studies suggest that exogenous application of PAP enhances plant systemic resistance to TMV infection in N. benthamiana [16]. To further investigate that the PAP plays an important role in the response to virus invasion, N. benthamiana leaves that overexpressed PAP-II gene (35S: PAP-II) was generated through Agrobacterium-mediated transient expression.

Figure 2. The overexpression of PAP-II in N. benthamiana plants was performed by Agrobacterium-mediated transient expression. (A) Overexpression vector of 35S: PAP-II. The complete open reading frame (ORF) of PAP-II was cloned into the binary vector pBI121 at XbaI and BamHI sites under the control of the CaMV35S promoter and nos terminator. (B) Western blotting analysis of PAP-II protein levels in the 35S: PAP-II and control (35S: 00) plants 48 h after agro-infiltration. The 35S: 00 or 35S: PAP-II-inoculated leaves were used for detection. Approximately 3 μg of protein from each sample was loaded onto the gels. Rubisco proteins were used as loading controls and stained with Ponceau S. The experiments were repeated three times with similar results.

In order to express PAP-II in N. benthamiana, Agrobacterium GV3101 containing 35S: PAP-II was used to infiltrate N. benthamiana leaves. The expression of the PAP-II protein in the agroinfiltrated leaves was confirmed by western blotting (Figure 2B). The PAP-II protein was detected at significantly higher level in the leaves of 35S: PAP-II plants compared with the control (35S: 00) plants after infiltration 48 h (Figure 2B).

Viral accumulation in 35S: PAP-II-overexpressing plants

Then, the 35S: PAP-II-overexpressing plants were inoculated with TMV-GFP and monitored for the induction of a resistanceresponse or virus spread for at least two weeks. To further confirm the role of PAP against virus infection, the levels of virus accumulation were detected over a 15-day time coursein 35S: PAP-II-overexpressing N. benthamiana leaves. Systemicsymptoms emerged in the second leaf above the inoculated leaves and spread further upper leaves. Virus accumulation was confirmed by direct observation of GFP fluorescence (Figure 3A), as well as by quantitative real time polymerase chainreaction (PCR) analysis of viral RNA (Figure 3B). Overexpression of PAP-II in N. benthamiana plants yielded a significantreduction in the GFP fluorescence in the noninoculated upper leaves (Figure 3A). In order to further confirm the results of GFP fluorescence, qRT-PCR was used to detect the level of viral RNA in molecular level. As expected, qRT-PCR analysis showedthat TMV accumulation levels were significantly reduced in the systemic leaves of 35S: PAP-II-overexpressing N. benthamianaplants compared with the levels observed in control (35S: 00) plants (Figure 3B). PAP is a highly toxic RIP produced by thepokeweed cells and exported outside the cells once synthesized [27]. And they possess pronounced antiviral properties [28]. Previous studies suggested that PAP exhibited antiviral activity against plant and animal viruses [29]. For example, the interactions between PAP and turnip mosaic virus genome linked protein (VPg) was investigated [13]. Our previous resultssuggest that exogenous application of PAP enhances N. benthamiana systemic resistance to TMV infection by regulating ROS levels. Furthermore, previous studies suggest that a RIP called α-MMC (alpha-momorcharin) had a broad-spectrum antiviral activity against phytopathogenic viruses [15]. Application of α-MMC can enhance the expression of SA-related genes, which strongly indicates that the plant systemic resistance can be activated by α-MMC [15]. Therefore, our studies suggest that RIP play an important role in plant systemic resistance against virus infection. However, investigation the role of RIP against virus infection by overexpression RIP in plants is rarely reported. Therefore, in this study, in order to further demonstrate the role of PAP against virus infection, N. benthamiana leaves that overexpressed PAP-II gene (35S: PAP-II) was generated. Overall, we conclude that the overexpression of PAP-II enhanced the plant systemic resistance to virus infection.

In conclusion, we have shown that overexpression of PAP-II increases plant systemic resistance against virus infection. Therefore, it may be a candidate to be used to develop virus resistant crop plants such as rice, tobacco, cotton and tomato.

Acknowledgements

We thank Professor David Baulcombe (the University of Cambridge) for providing TMV-GFP. This work was supported by the National Natural Science Foundation of China (Grant no. 31500209), Natural Science Foundation of the Higher EducationInstitutions of Jiangsu Province of China (Grant no. 15KJB210007) and Natural Science Foundation of Yangzhou (Grant no. YZ2015106).